A 125-kDa protein that inhibits phospholipase D (PLD) activity stimulated by ADP-ribosylation factor and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] was purified from rat brain cytosol by sequential chromatographies on DEAE Sephacel, phenyl HPLC, heparin-5PW HPLC, and gel filtration HPLC columns. The purified PLD inhibitor was identified as amphiphysin I by peptide sequencing and immunoblot analysis with antibodeis to amphiphysin. Purified amphiphysin I inhibited both human PLD1(hPLD1) and mouse PLD2(mPLD2) with an IC50 value of 10 nM. A bacterially expressed fusion protein, glutathione S-transferase(GST)-amphiphysin, also inhibited hPLD1 and PLD2 with a potency similar to that of brain amphiphysin.. Thge inhibitory effect of amphiphysin I appeared to be the result of direct interaction between amphiphysin and PLD because PLD could be precipitated with GST-amphiphysin. Transient expression of human amphiphysin I in Cos-7 cells resulted in a 70% inhibition of PLD activity measured after stimulation with a phorbol ester. Similar extents of inhibition could be demonstrated with Cos-7 cells cotransfected with amphiphysin and hPLD1 or mPLD2. We also showed that the 80-kDa amphiphysin II inhibited the activities of PD1 and PLD2 both in vitro and in vivo and associated with them. Amphiphysin I is a SH3 domain-containing protein that has been implicated in synaptic vesicle endocytosis in the brain by interacting with dynamin through SH3 domain. We thus propose that the amphiphysin-dependent inhibition of PLD might play a regulatory role in the formation of coated vesicles.